Phylogenomics of the genus Hypoxylon based on 50 new high quality genomes and with special emphasis on the H. rubiginosum complex

摘要：利用综合方法（包括仔细的形态学研究、利用HPLC/DAD-MS生成次生代谢物图谱和多位点分子系统发育）对Hypoxylon属（Hypoxylon科，Xylariales）及其相关属进行了大量的分类研究。此外，从这些真菌中发现了一些前所未有的次生代谢物，其中许多是化学分类标记和/或显示出很强的生物活性。在目前正在进行的项目中，共生成了13个来自木杉科（Hypoxylaceae）和木杉属（Xylaria hypoxylon）物种的基因组序列，并对其进行了次生代谢产物生物合成基因簇（BGC）的注释，发现了600多个BGC，这些BGC主要局限于单个物种。此外，各种基因组比较方法（ANI-平均核苷酸身份；AAI-平均氨基酸身份；pocp -保守蛋白百分比）首次在真菌中大规模应用。此外，首次实现了Sordariomycetes在家族水平上的系统基因组学方法，基于全基因组序列的系统发育结果广泛符合多位点系统发育。该数据矩阵由同时构建的基于HPLC- dad /MS的化合物文库补充，该文库包含测序菌株提取物的HPLC图谱。该文库包含超过100种来自木氧根科的纯次生代谢物，因此非常适合于去复制（即，通过在标准化的HPLC系统中比较其光谱和保留时间来对已知代谢物进行早期鉴定）。后续项目旨在着手这些先决条件，针对rubiginosum Hypoxylon复合体，该复合体由许多内生菌和明显罕见的宿主特异性腐养菌以及生长在被子植物木材上的无处不在的子囊菌组成。总共将选出50个具有代表性的菌株。一些在系统发育上与子囊孢子衍生菌株密切相关的内生菌也将包括在内。该研究的目的是：a)生成和注释50个Hypoxylon属不同物种的基因组序列，重点是H. rubiginosum复合体；b)评估基因组比较参数（ANI， AAI， POCP）的效用，以确定物种边界；c)基于系统基因组方法稳定Hypoxylon的系统发育；D)研究同一物种内生和子囊孢子衍生菌株的基因组和次级代谢组；e)进一步研究近缘种BGC之间的进化关系；f)鉴定编码重要化学分类标记分子的BGC；g)发现和设计迄今未知的hypxylon及其BGC代谢物。

The genus Hypoxylon (Hypoxylaceae, Xylariales) and related genera have been the subject of intensive work regarding the evaluation of its taxonomy by polythetic methodology (comprising careful morphological studies, generation of secondary metabolite profiles by using HPLC/DAD-MS and multi-locus molecular phylogenies). Moreover, several unprecedented secondary metabolites were discovered from these fungi, many of those are chemotaxonomic markers and/or show strong bioactivities. In a currently ongoing project 13 genome sequences from species of the Hypoxylaceae and Xylaria hypoxylon were generated and annotated for secondary metabolite biosynthetic gene clusters (BGC), and over 600 BGC were found, which were mainly restricted to individual species. Furthermore, various methods for genome comparison (ANI- average nucleotide identity; AAI- average amino acid identity; POCP-percentage of conserved proteins), were firstly applied on a larger scale in fungi. In addition, a phylogenomic approach was realized at the family level for Sordariomycetes for the first time, and the resulting phylogeny based on the full genome sequences was found widely in accordance with the multi-locus phylogeny. This data matrix was complemented by a concurrently constructed HPLC-DAD/MS based compound library containing HPLC profiles of extracts from the strains that were sequenced. The library contains over 100 pure secondary metabolites from Hypoxylaceae and is therefore perfectly suited for dereplication (i.e., early stage identification of known metabolites by comparing their spectra and retention times in a standardized HPLC system. The follow-up project is intended to embark on these prerequisites, targeting the Hypoxylon rubiginosum complex, which comprises numerous endophytes and apparently rare, host-specific saprotrophs as well as ubiquitous ascomycetes that grow on angiosperm wood. In total, 50 representative strains of this complex will be selected. Some endophytes that appear phylogenetically closely related to the ascospore-derived strains will also be included. The strains will be subjected to genome sequencing and concurrently studied intensively for metabolite production The aims of the study will be: a) generate and annotate 50 genome sequences of various species of the genus Hypoxylon with emphasis on the H. rubiginosum complex; b) evaluate the utility of genome comparison parameters (ANI, AAI, POCP), to define species boundaries; c) stabilize the phylogeny of Hypoxylon based on phylogenomic approaches; d) study the genomes and secondary metabolome of endophytic vs. ascospore derived strains of the same species; e) further investigate evolutionary relationships between the BGC of closely related species; f) identify the BGC encoding some important chemotaxonomic marker molecules; and g) to discover and engineer hitherto unknown metabolites of Hypoxylon and their BGC.